Project description:Mutations in PHF8 are associated with X-linked mental retardationand cleft lip/cleft palate. PHF8 contains a plant homeodomain(PHD) in its N-terminus and is member of a family of JmjC-domaincontaining proteins. While PHDs can act as methyl lysine recognitionmotifs, JmjC-domains can catalyze lysine demethylation. Here,we show that PHF8 is a histone demethylase that removes repressivehistone H3 dimethyl lysine 9 marks. Our biochemical analysisrevealed specific association of the PHF8 PHD domain with histoneH3 trimethylated at lysine 4 (H3K4me3). Chromatin-immunoprecipitationfollowed by high throughput sequencing indicated that PHF8 isenriched at transcription start sites of many active or poisedgenes, mirroring the presence of RNA polymerase II (RNAPII)and of H3K4me3-bearing nucleosomes. We show that PHF8 can actas a transcriptional co-activator and its activation functionlargely depends on binding of the PHD to H3K4me3. Furthermore,we present evidence for direct interaction of PHF8 with theC-terminal domain of RNAPII. Importantly, a PHF8 disease mutantis defective in demethylation and in co-activation. This isthe first demonstration of a chromatin-modifying enzyme whichis globally recruited to promoters through its association withH3K4me3 and RNAPII. This SuperSeries is composed of the following subset Series: GSE20563: Knockdown of PHF8 in HeLa S3 cells GSE20725: ChIP-Seq of PHF8 and H3K4me3 Refer to individual Series
Project description:PHF8 is an H3K9me2 demethylase, interacts with H3K4me3 and RNA Polymerase II, is enriched at thousands of transcription start sites and can act as a transcriptional co-activator. In the following experiment we wanted to analyze the impact of PHF8-knockdown on transcript levels in HeLa cells. Three independent replicate knockdown experiments with PHF8-shA and control NT-sh were performed (6 samples in total). The two samples of each replicate experiment were hybridized in two color dye swap experiments (6 arrays in total).
Project description:PHF8 is an H3K9me2 demethylase, interacts with H3K4me3 and RNA Polymerase II, is enriched at thousands of transcription start sites and can act as a transcriptional co-activator. In the following experiment we wanted to analyze the impact of PHF8-knockdown on transcript levels in HeLa cells.
Project description:While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here, we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while utilizing multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1/S transition in conjunction with E2F1, HCF-1 and Set1A, at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the Condensin II loading process. Accordingly, the HEAT repeat clusters in two non-SMC Condensin II subunits, N-CAPD3 and N-CAPG2, are capable of recognizing H4K20me1, and ChIP-seq. analysis demonstrate a significant overlap of Condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression. unsynchronized HeLa cells were used to profile H3K4me2 and E2F1; unsynchronized or G1 or G1/S synchronized HeLa cells were used to profile PHF8; M phase synchronized HeLa cells were used to profile SMC4 and H4K20me1